PROJECT SUMMARY Safety pharmacology concerns are a major cause of drug attrition during development, with cardiac arrhythmia accounting for the majority of cases. Current preclinical screening assays are incapable of robustly predicting arrhythmogenic potential in new drugs due to their inability to accurately model human myocardial function. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in future drug efficacy/toxicity screening applications. However, variability in differentiation and functional maturation rates is a well-established phenomenon in hPSC cultures, and there currently exists no standardized assay for determining appropriate levels of hPSC-CM functional performance. The widespread adoption of hPSC-CMs in preclinical screening applications is predicated on an ability to adequately define maturation end points and acceptable levels of functional parameters for different lines, so that data obtained using various cell sources can be reliably compared. To meet this unmet need, we will establish a nanopatterned microelectrode array (nanoMEA)-based functional phenotyping assay for drug- induced cardiotoxicity screening. Our developed assay will determine 1) whether inherent differences in the degree of maturation, functional performance, and pharmacologic response exist between cardiomyocytes derived from different PSC lines, and 2) whether this variability impacts these cell?s suitability for use in preclinical assays. Analysis of this variance will be used to propose a set of standardized criteria incorporating minimum acceptable performance levels for hPSC-CMs prior to their selection for use in more widespread preclinical applications. Working towards this, we will first optimize and validate our prototype multi-well nanoMEA platform for high- throughput, high-content screening of baseline hPSC-CM electrophysiological properties (Aim 1.1). We will then utilize this platform in conjunction with transcriptome analysis to screen and determine optimal maturation strategies for hPSC-CM cultures (Aim 1.2). We will also screen multiple embryonic and induced stem cell lines to determine the level of functional variability present in cardiomyocytes derived from different hPSC sources (Aim 2.1). Finally, we will demonstrate the predictive capacity of our nanoMEA-based functional assay in terms of accurately detecting arrhythmogenic potential against a panel of known arrhythmogenic and non- arrhythmogenic compounds (Aim 2.2). This research is of high significance due to its potential to yield both fundamental knowledge of mechanisms of cardiac maturation, as well as providing validation of a powerful new tool with which to perform subsequent preclinical screening.